EP3818074A1 - Saccharomyces cerevisiae strains expressing glucoamylase enzymes and exogenous xylanase and their use in the production of bioethanol - Google Patents
Saccharomyces cerevisiae strains expressing glucoamylase enzymes and exogenous xylanase and their use in the production of bioethanolInfo
- Publication number
- EP3818074A1 EP3818074A1 EP19758897.3A EP19758897A EP3818074A1 EP 3818074 A1 EP3818074 A1 EP 3818074A1 EP 19758897 A EP19758897 A EP 19758897A EP 3818074 A1 EP3818074 A1 EP 3818074A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- saccharomyces cerevisiae
- glucoamylase
- xylanase
- yeast
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to yeast strains of Saccharomyces cerevisiae genetically modified so as to co-express genes encoding glucoamylases of fungal origin and of Saccharomyces cerevisiae var. diastaticus, and a gene encoding a fungal xylanase.
- Such strains find, in particular, application in the production of biofuel, in particular bioethanol.
- the present invention also relates to a process for obtaining these yeasts and to the use of these yeasts in the production of bioethanol.
- the so-called first generation bioethanol is produced by fermentation of hexoses (six-carbon sugars) contained in biomass rich in starch (corn kernels, barley, wheat, cassava, potato tubers, etc.) or in sucrose (cane sugar, sugar beet, sugar sorghum etc.), while so-called second generation bioethanol is generated by the transformation of cellulose and hemicellulose contained in agricultural residues such as cereal straws, corn stover, residues forest, wood, energy crops such as switchgrass or short or very short rotation coppice (poplar for example).
- the industrial preparation includes the use of Saccharomyces cerevisiae yeast strains, which allow the glucose of the biomass to be fermented into ethanol with an alcoholic titer, high productivity and yield.
- the process for converting starch to bioethanol includes pre-hydrolysis and liquefaction of starch from biomass, conversion of liquefied starch to fermentable sugars (by hydrolysis of starch), and fermentation of these sugars in ethanol - these last two stages are often carried out simultaneously.
- the hydrolysis of starch requires the action of so-called amylolytic enzymes.
- Saccharomyces cerevisiae yeasts are generally devoid of this type of enzyme
- the production of ethanol from biomass composed of starch is carried out in two stages: a first stage of adding amylolytic enzymes to the biomass so as to pre-hydrolyze and liquefy the starch contained in the biomass, and a second step where other enzymes (amylolytic enzymes, enzyme cocktails, proteases, and / or trehalase, etc.) are used to hydrolyze the starch liquefied and a strain of Saccharomyces cerevisiae to ferment the fermentable sugars thus released.
- enzymes amylolytic enzymes, enzyme cocktails, proteases, and / or trehalase, etc.
- Saccharomyces cerevisiae comprising, integrated into their genome, exogenous glucoamylase genes.
- These genetically modified Saccharomyces cerevisiae strains allow partial hydrolysis of liquefied starch and alcoholic fermentation to be carried out simultaneously (WO 2017/037362).
- WO 2017/037362 Despite the increased performance of the modified Saccharomyces cerevisiae strains, there is still a need for new and improved yeast strains for the production of first generation ethanol.
- the present invention relates to Saccharomyces cerevisiae yeast strains which have improved properties compared to specialized yeast strains commonly used in the production of first generation bioethanol, and also compared to Saccharomyces cerevisiae strains comprising, integrated into their genome, exogenous glucoamylase genes (WO 2017/037362). More specifically, the inventors of the present invention have developed a genetically modified strain of Saccharomyces cerevisiae, said strain co-expressing several heterologous glucoamylase genes, and a heterologous xylanase gene.
- Saccharomyces cerevisiae strains according to the invention coexpress both a gene encoding a glucoamylase of fungal origin and a gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus, as well as a xylanase gene of fungal origin.
- the inventors have demonstrated that these strains are capable of hydrolyzing the liquefied starch extracted from the biomass while successfully fermenting the sugars resulting from this hydrolysis.
- the use of a yeast strain according to the present invention makes it possible to replace all or part of the quantity of exogenous enzymes necessary during the conversion of the liquefied starch into bioethanol and produces bioethanol with a higher yield. that the strains known in the art.
- the present invention relates to a yeast strain of Saccharomyces cerevisiae, characterized in that it co-expresses:
- the fungal xylanase is an Aspergillus niger xylanase or a Trichoderma reesei xylanase.
- the xylanase of fungal origin can be an AspergiUus niger xylanase which is coded by the nucleic sequence SEQ ID NO: 5 or which consists of the polypeptide sequence SEQ ID NO: 6 or a functional variant of the polypeptide sequence SEQ ID NO: 6.
- the xylanase of fungal origin can be a Trichoderma reesei xylanase which is coded by the nucleic sequence SEQ ID NO: 7 or which consists of the polypeptide sequence SEQ ID NO: 8 or a functional variant of the polypeptide sequence SEQ ID NO: 8.
- the glucoamylase of Saccharomyces cerevisiae var. diastaticus is encoded by the nucleic sequence SEQ ID NO: 3 or consists of the polypeptide sequence SEQ ID NO: 4 or a functional variant of the polypeptide sequence SEQ ID NO: 4.
- the glucoamylase of fungal origin is chosen from the group consisting of: a glucoamylase from Aspergillus niger, a glucoamylase from Saccharomycopsis fibuligera, a glucoamylase from Trichoderma reesei, a glucoamylase from Thermomyces lanuginosis, a glucoamylase from Rhiz and an Aspergillus oryzae glucoamylase.
- the fungal glucoamylase can be an Aspergillus niger glucoamylase which is coded by the nucleic sequence SEQ ID NO: 1 or which consists of the polypeptide sequence SEQ ID NO: 2 or a functional variant of the polypeptide sequence SEQ ID NO: 2.
- the yeast strain of Saccharomyces cerevisiae according to the invention comprises:
- n is an integer between 2 and 10
- p is an integer between 2 and 10.
- m is 1 or 4.
- n 6 and p is 4.
- the gene encoding fungal xylanase, the gene encoding fungal glucoamylase, and the gene encoding Saccharomyces cerevisiae var glucoamylase. diastaticus are integrated within the genome of the yeast strain of Saccharomyces cerevisiae according to the invention.
- the yeast strain of Saccharomyces cerevisiae according to the invention is the strain deposited on April 26, 2017 at the CNCM under the number 1-5201.
- the present invention relates to a method for obtaining a yeast strain of Saccharomyces cerevisiae useful for the production of bioethanol, said method comprising the steps consisting in:
- step (b) culture and ferment the yeast obtained in step (a) on a synthetic dextrin medium;
- the present invention relates to a method for increasing the yield of bioethanol production of a yeast strain of Saccharomyces cerevisiae, said method comprising the steps consisting in:
- step (b) genetically modifying the yeast of step (a) so that it additionally expresses a gene coding for a xylanase of fungal origin;
- step (c) culture and ferment the yeast obtained in step (b) on a synthetic dextrin medium
- the method for increasing the yield of bioethanol production of a yeast strain of Saccharomyces cerevisiae is characterized in that the yeast of Saccharomyces cerevisiae of step (a) is the yeast strain of Saccharomyces cerevisiae filed on July 9, 2015 at the CNCM under number 1-4997.
- the present invention relates to a method for producing bioethanol from a biomass, said biomass method comprising the steps consisting in:
- step (b) reacting the liquefied starch obtained in step (a) with a strain of yeast
- Saccharomyces cerevisiae for producing bioethanol
- step (c) extract the bioethanol produced in step (b).
- the present invention also relates to the use of a yeast strain of Saccharomyces cerevisiae disclosed here, for the production of bioethanol.
- the present invention relates to yeast strains Saccharomyces cerevisiae having a high yield in production of first generation bioethanol, in particular bioethanol produced from biomass comprising starch.
- yeast strain refers to a relatively homogeneous population of yeast cells.
- a yeast strain is obtained from a clone, a clone being a population of cells obtained from a single yeast cell.
- a starting strain of Saccharomyces cerevisiae yeast is any strain of Saccharomyces cerevisiae which can be genetically modified to introduce the heterologous xylanase and glucoamylase genes according to the invention.
- the starting Saccharomyces cerevisiae strain is a strain known to be useful in the production of bioethanol, such as, for example, the yeasts of Saccharomyces cerevisiae used by the first generation ethanol producers, which are yeasts specialized to optimize the profitability of the production process.
- yeasts which are well known in the art, include: Ethanol Red ® (LEAF), Thermosacc ® (Lallemand), Angel Super Alcohol ® (Angel) and Fali ® (AB Mauri).
- the expected qualities of these yeasts are their capacity to rapidly produce high concentrations of ethanol and to exhaust the sugars from the fermentation media over the temperature and pH ranges representative of industrial conditions.
- the strains according to the present invention are improved strains compared to the Saccharomyces cerevisiae yeast strains previously developed by the present inventors, that is to say compared to Saccharomyces cerevisiae yeast strains where the introduction of a gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus and a gene encoding a fungal glucoamylase had made it possible to obtain strains with excellent capacities for starch hydrolysis (WO 2017/037362).
- glucoamylase an amino acid sequence which, when expressed, results in the formation of a functional glucoamylase protein.
- glucoamylase is meant here an enzyme capable of hydrolyzing the ⁇ -1,4 glycosidic bonds of raw or soluble starch from the non-reducing end of amylose and amylopectin.
- Amylases are also known by the name of amyloglucosidases or g-amylases (Medline reference: EC 3.2.1.3).
- glucoamylase is capable of slowly hydrolyzing the ⁇ -1,6 bonds of amylopectin molecules, provided that the neighboring bond in the sequence is a bond a- 1,4.
- fungal glucoamylase designates any glucoamylase originating from a fungus (fungus or fungus) and whose corresponding gene can be integrated into the genome of a yeast strain so that the expression of the gene results in the formation of a functional glucoamylase protein.
- a glucoamylase of fungal origin can be chosen from commercial glucoamylases known for their good enzymatic activity.
- a fungal glucoamylase can be selected from the group consisting of: a glucoamylase from Aspergillus niger, a glucoamylase from Saccharomycopsis fibuligera, a glucoamylase from Trichoderma reesei, a glucoamylase from Rhizopus oryzae, a glucoamylase of "Aspergillus oryzae and a glucoamylase Thermomyces lanuginosis.
- glucoamylases are known to those skilled in the art, and their sequences are accessible under the following GenBank references (www.ncbi.nlm.nih.gov/genbank/): Trichoderma reesei (ETS06561 ), Rhizopus oryzae (BAA00033), Aspergillus oryzae (BAA00841), Thermomyces lanuginosus (ABQ23180).
- the glucoamylase of fungal origin is a glucoamylase from Aspergillus niger or from Saccharomycopsis fibuligera.
- the glucoamylase of Aspergillus niger is coded by the GLAA gene which has the nucleic sequence SEQ ID NO: 1, and has the protein sequence the sequence SEQ ID NO: 2.
- the glucoamylase of Saccharomycopsis fibuligera is coded by the gene GLU0111 which has the nucleic sequence SEQ ID NO: 9, and has the protein sequence sequence SEQ ID NO: 10.
- the glucoamylase of Saccharomyces cerevisiae var. diastaticus is encoded by the STA1 gene which has the nucleic sequence SEQ ID NO: 3, and has the protein sequence the sequence SEQ ID NO: 4.
- a yeast strain of Saccharomyces cerevisiae according to the invention is characterized in that it co-expresses:
- such a strain of Saccharomyces cerevisiae can in particular be characterized in that it contains the nucleic sequence SEQ ID NO: 1 and the nucleic sequence SEQ ID NO: 3.
- such a strain of Saccharomyces cerevisiae can in particular be characterized in that the glucoamylase of Saccharomyces cerevisiae var. diastaticus has the protein sequence SEQ ID NO: 4 and the glucoamylase of Aspergillus niger has the protein sequence SEQ ID NO: 2.
- a yeast strain of Saccharomyces cerevisiae according to the invention differs from the strain of Saccharomyces cerevisiae previously developed by the present inventors (WO 2017/037362) in that in addition to the gene encoding a glucoamylase of fungal origin; and the gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus, it also co-expresses a gene coding for a xylanase of fungal origin.
- xylanase an amino acid sequence which, when expressed, results in the formation of a functional xylanase protein.
- xylanase is meant here a glycoside hydrolase enzyme which hydrolyzes the (l 4) -PD-xylosidic bonds in xylans, thus generating xylose.
- Xylanases are also known by the name of endo- 1, 4- -xylanases (Medline reference: EC 3.2.1.8). These enzymes are involved in the breakdown of hemicellulose, one of the main constituents of cell walls in plants. They are produced in particular by fungi, bacteria, yeasts, seaweed, protozoa, snails, crustaceans, insects and certain seeds, but not by mammals.
- xylanase is a xylanase of fungal origin.
- xylanase of fungal origin designates any xylanase originating from a fungus (fungus or fungus) and the corresponding gene of which can be integrated into the genome of a yeast strain so that the expression of the gene results in the formation of a functional xylanase protein.
- a xylanase of fungal origin can be chosen from commercial xylanases known for their good enzymatic activity.
- a fungal xylanase can be selected from the group consisting of: an Aspergillus niger xylanase, an Aspergillus awamori xylanase, an Aspergillus tubingensis xylanase, an Aspergillus nidulans xylanase , and a Trichoderma reesei xylanase.
- GenBank references www.ncbi.nlm.nih.gov/genbank/: Aspergillus niger (FJ785738), Aspergillus awamori (X78115), Aspergillus tubingensis (L26988), Aspergillus nidulans (Z49892), and Trichoderma reesei (X69573).
- the xylanase of fungal origin is a Aspergillus niger or Trichoderma reesei xylanase.
- the Aspergillus niger xylanase is encoded by the XYN1 gene which has the consensus nucleic sequence SEQ ID NO: 5, and has the protein sequence the sequence SEQ ID NO: 6.
- Trichoderma reesei xylanase is encoded by the XYN2 gene which has the consensus nucleic sequence SEQ ID NO: 7, and has the protein sequence the sequence SEQ ID NO: 8.
- fungal glucoamylase and “Saccharomyces cerevisiae var. diastaticus "should not be interpreted strictly: they include the glucoamylases of fungal origin and of Saccharomyces cerevisiae var. diastaticus which are coded by the nucleic sequences as described above, but also the functional variants of these glucoamylases.
- xylanase of fungal origin encompasses xylanases of fungal origin which are encoded by the nucleic sequences as described above, but also the functional variants of these xylanases.
- a functional variant of a glucoamylase or of a xylanase according to the invention has a protein sequence having a percentage identity of at least 80%, 90%, or 95%, more particularly 99%, with the protein sequence of said glucoamylase or xylanase, respectively.
- the functional variants of the glucoamylase of Aspergillus niger have a protein sequence exhibiting a percentage identity of at least 80%, at least 90%, or at least 95%, more particularly at least minus 99%, with the sequence SEQ ID NO: 2; functional variants of the glucoamylase of Saccharomyces cerevisiae var.
- diastaticus have a protein sequence having a percentage identity of at least 80%, at least 90%, or at least 95%, more particularly 99%, with the sequence SEQ ID NO: 4; the functional variants of the Aspergillus niger xylanase have a protein sequence having a percentage identity of at least 80%, at least 90%, or at least 95%, more particularly 99%, with the sequence SEQ ID NO: 6; and the functional variants of Trichoderma reesei xylanase have a protein sequence having a percentage identity of at least 80%, at least 90%, or at least 95%, more particularly 99%, with the sequence SEQ ID NO: 8.
- Percent Identity is a comparison between amino acid sequences, and is determined by comparing two optimally aligned sequences on a comparison window. A person skilled in the art knows how to calculate a percentage of identity between two sequences and has many tools allowing this. One of the two sequences can have amino acid insertions, substitutions and deletions compared to the other sequence.
- glucoamylase a variant which retains the enzymatic activity of glucoamylase and this with similar kinetics of starch hydrolysis.
- functional variant of a xylanase a variant which retains the enzymatic activity of the xylanase, and this with similar kinetics of xylan hydrolysis.
- the strains according to the invention can be generated by any suitable method.
- Those skilled in the art know, for example, multiple methods allowing to introduce a gene into a yeast strain, in particular via the use of vectors comprising expression cassettes.
- the term “vector” is intended to mean any DNA sequence into which it is possible to insert fragments of foreign nucleic acids, the vectors making it possible to introduce foreign DNA into a host cell.
- Examples of vectors are plasmids, cosmids, vectors derived from viruses.
- the vectors allow either the integration of heterologous genes directly into the yeast genome, or their expression in an independent plasmid.
- the introduction of vectors into a host cell is a process widely known to those skilled in the art. Several methods are notably described in “Current Protocols in Molecular Biology”, 13.7.1-13.7.10; or in Ellis et al., Intégrative Biology, 2011, 3 (2), 109-118.
- a strain of Saccharomyces cerevisiae according to the invention is prepared from a strain of Saccharomyces cerevisiae previously developed by the present inventors and described in WO2017 / 037362 (that is to say say from a strain already containing a gene coding for a glucoamylase of fungal origin and a gene coding for a glucoamylase from Saccharomyces cerevisiae var. diastaticus).
- a strain of Saccharomyces cerevisiae according to the invention is prepared by integrating the three genes into a strain of Saccharomyces cerevisiae such as, for example, a specialized strain - see above.
- the gene encoding a glucoamylase of fungal origin, the gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus, and the gene encoding a xylanase of fungal origin can be inserted within a single vector, or within two or three separate vectors.
- a suitable vector can be a plasmid.
- a vector used to introduce a gene into a strain of Saccharomyces cerevisiae may contain a selection marker.
- selection marker is intended to mean a gene the expression of which gives the yeasts which contain it a characteristic which makes it possible to select them. It may for example be an antibiotic resistance gene or a gene allowing the growth of yeast in a particular medium.
- a gene (glucoamylase or xylanase) according to the invention is generally operably linked to a promoter, a terminator and / or any other sequence necessary for its expression in yeast.
- the terms “operationally linked” and “operably linked” are used interchangeably and refer to a functional link between the elements allowing the expression of the gene and possibly its regulation (regulatory sequences 5 ′ and 3 ′) and the sequence of the reporter gene that they control.
- a person skilled in the art knows how to select the promoters, terminators and other sequences necessary for the expression of a gene in a yeast of Saccharomyces cerevisiae.
- the expression of a gene is controlled by a so-called “strong” promoter (that is to say a promoter having a transcriptional potential high so that the gene is strongly expressed).
- a strong promoter is for example the pADH1 promoter, the pTEF promoter, or the pTDH3 promoter.
- a yeast strain of Saccharomyces cerevisiae according to the invention can comprise several copies of at least one of the glucoamylase genes of fungal origin, of Saccharomyces cerevisiae var. diastaticus, and xylanase of fungal origin.
- a strain of Saccharomyces cerevisiae according to the invention comprises, m copies of the gene encoding the xylanase of fungal origin; n copies of the gene encoding the fungal glucoamylase; and p copies of the gene encoding the glucoamylase of Saccharomyces cerevisiae var.
- n and p can independently be equal to 2, 3 , 4, 5, 6, 7, 8, 9, or 10 and m can be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
- m is equal to 1 or 4.
- n is equal to 6 and p is equal to 4.
- the invention relates in particular to the yeast strain of Saccharomyces cerevisiae which has been deposited, by the present Applicant, at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25-28 rue du Do Budapest Roux, 75 724 Paris Cedex 15 ), under the Budapest Treaty, under number 1-5201 on April 26, 2017.
- This strain comprises 6 copies of the glucoamylase gene from AspergiUus niger, 4 copies of the glucoamylase gene from Saccharomyces cerevisiae var. diastaticus and 1 copy of the Aspergillus niger xylanase gene.
- II Method for obtaining Genetically Modified Saccharomyces cerevisiae Strains Useful for the Production of Bioethanol
- the present inventors have also developed a method for obtaining strains of Saccharomyces cerevisiae useful in the production of bioethanol.
- the method includes the steps of:
- step (b) culture and ferment the yeast from step (a) on a synthetic dextrin medium
- (c) select at least one strain exhibiting fermentation kinetics in the synthetic dextrin medium at least equal to or greater than the fermentation kinetics of the strain of Saccharomyces cerevisiae deposited on July 9, 2015 at the CNCM under number 1-4997.
- step (a) of genetic modification it is preferable to select the clones having correctly integrated the genes introduced.
- the yeast from step (a) which is used in step (b) is a clone having correctly integrated the gene coding for xylanase of fungal origin, the gene coding for glucoamylase of fungal origin, and the gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus.
- Those skilled in the art know how to select such clones, for example by using a selection marker introduced into the starting Saccharomyces cerevisiae yeast.
- the Examples provided in this document describe an example of a method for selecting clones.
- a clone obtained in step (a) is cultured in a medium rich (in nutrients, medium type YPG), then the culture supernatant is transferred to a minimum medium containing xylan as carbon source.
- a second yeast the CelluX TM yeast (which was deposited by the Applicant on December 12, 2013 at the CNCM under the number 1-4829), is added to the reaction medium.
- a clone is selected (that is to say identified as having correctly integrated the introduced genes), if there is growth of the CelluX TM strain.
- the yeast strain of Saccharomyces cerevisiae obtained in step (a) of the method described above is a strain according to the present invention. Its characteristics are therefore identical to those described in the previous section.
- synthetic dextrin medium is understood here to mean a cell culture medium, preferably a culture medium for yeast cells, containing dextrins, as known to those skilled in the art. It is for example a culture medium containing starch dextrins (220 g / kg), yeast extract (5 g / kg), urea (2 g / kg), potassium dihydrogen phosphate (1 g / kg) as well as minerals and vitamins (such as vitamin B1 and vitamin B6).
- step (c) the selection of an efficient and useful strain of Saccharomyces cerevisiae in the production of bioethanol is done by comparing its fermentation kinetics with that of the strain of Saccharomyces cerevisiae deposited by the present Applicant on 9 July 2015 at the CNCM under the number 1-4997.
- the kinetics of fermentation can be easily measured by various techniques known to those skilled in the art. For example, the fermentation kinetics can be measured via fermentation monitoring by weighing over time.
- the strain 1-4997 which serves as a reference, is one of the strains previously developed by the present inventors and described in WO 2017/037362.
- the strain of Saccharomyces cerevisiae 1-4997 comprises at least 4 copies of the gene coding for glucoamylase from Aspergillus niger and at least 3 copies of the gene coding for glucoamylase from Saccharomyces cerevisiae var. diastaticus.
- a strain selected by a method according to the present invention therefore necessarily has identical or superior fermentation properties to the strain Saccharomyces cerevisiae 1-4997, in a synthetic dextrin medium. Consequently, the present inventors have also developed a method making it possible to increase the yield of bioethanol production of a yeast strain of Saccharomyces cerevisiae. III - Method to Increase the Production Yield in Bioethanol of a Saccharomyces cerevisiae Strains
- the present invention therefore also relates to a method making it possible to increase the yield of bioethanol production of a yeast strain of Saccharomyces cerevisiae, said method comprising the steps consisting in:
- step (b) genetically modifying the yeast of step (a) so that it additionally expresses a gene coding for a xylanase of fungal origin;
- step (c) culture and ferment the yeast obtained in step (b) on a synthetic dextrin medium
- the strain of step (a) can be any strain of Saccharomyces cerevisiae co-expressing a gene encoding a glucoamylase of fungal origin, and a gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus.
- the glucoamylase of fungal origin is selected from the group consisting of: a glucoamylase from Aspergillus niger, a glucoamylase from Saccharomycopsis fibuligera, a glucoamylase from Trichoderma reesei, a glucoamylase from Rhizopus oryzae, a glucoamylase from " oryzae and a glucoamylase Thermomyces lanuginosis as described above
- the fungal glucoamylase whose exogenous gene is present in the strain of Saccharomyces cerevisiae of step (a) is a glucoamylase from Aspergillus niger , for example an Aspergillus niger glucoamylase encoded by the nucleic sequence SEQ ID NO: 1 or an Aspergillus niger glucoamylase consisting of the polypeptide sequence S
- the glucoamylase of Saccharomyces cerevisiae var. diasta ticus whose exogenous gene is present in the strain of Saccharomyces cerevisiae of step (a) is a glucoamylase from Saccharomyces cerevisiae var. diastaticus encoded by the nucleic sequence SEQ ID NO: 3 or a glucoamylase from Saccharomyces cerevisiae var. diastaticus consisting of the polypeptide sequence SEQ ID NO: 4 or a functional variant of the polypeptide sequence SEQ ID NO: 4.
- the strain of step (a) is a strain of Saccharomyces cerevisiae co-expressing a gene encoding a glucoamylase of fungal origin, and a gene encoding the glucoamylase of Saccharomyces cerevisiae var. diastaticus, as described in WO 2017/037362.
- the strain of step (a) is the yeast strain of Saccharomyces cerevisiae deposited, by the present Applicant, on July 9, 2015 at the CNCM under the number 1-4997.
- Step (b) of genetic modification can be carried out by any method known to a person skilled in the art, as already indicated above.
- a selection can be made of the clones having correctly integrated the gene coding for the xylanase of fungal origin, for example by using a selection marker.
- Steps (c) and (d) of the method making it possible to increase the yield of bioethanol production of a yeast strain of Saccharomyces cerevisiae can be carried out as indicated for the selection method according to the invention.
- the invention also relates to any yeast strain obtained by a method of selection or increase in yield according to the invention.
- the invention also relates to a yeast obtained by culture of one of the strains of the invention.
- the methods for cultivating a yeast strain are known in the art, and those skilled in the art know how to optimize the culture conditions for each strain according to its nature.
- the yeast strains of the invention and the yeasts obtained by culture of these strains are particularly advantageous for producing bioethanol from biomass, in particular from biomass containing starch.
- the present invention therefore relates to the use of a strain of Saccharomyces cerevisiae according to the present invention for the production of bioethanol from a biomass containing starch.
- the present invention also relates to a method for producing bioethanol from a biomass containing starch, said method comprising the steps consisting in: (a) prehydrolyzing (i.e. partially hydrolyzing) and liquefying the starch from the biomass;
- step (b) reacting the biomass containing the pre-hydrolyzed and liquefied starch obtained in step (a) with a yeast strain of Saccharomyces cerevisiae according to the present invention to produce bioethanol; and
- biomass refers to all organic matter of plant origin that can become a source of energy after transformation.
- the biomass comes from agricultural or agro-food products and / or co-products.
- a biomass can come from corn, wheat, barley, rye, sorghum, cassava, triticale, potato, sweet potato, sugar cane, sugar beet, sugar sorghum.
- the biomass contains starch.
- the starch-rich biomasses can be chosen, or come from, for example, corn kernels, barley, wheat, cassava, potato tubers, etc.
- Steps (a), (b) and (c) of the method according to the invention can be carried out as in the case of a conventional process for producing bioethanol. Such steps are known to those skilled in the art.
- the invention is particularly applicable to the production of bioethanol as a fuel, but also to the production of bioethanol for the food, chemical, pharmaceutical and cosmetic industries.
- Figure 1 (A) Loss of mass observed for transformants of the strain deposited at the CNCM under the number 1-4997 during fermentation in the “Alcohol Max” medium. (B) Loss of mass observed for the transformants of strain number 1-4997 during fermentation in the “Dextrin” medium.
- Figure 2 (A) Comparison of the fermentation kinetics of the different strains studied. (B) Comparison of the values of ethanol contents, glycerol / ethanol mass ratios as well as ethanol yields calculated on the glucose potential of the substrate used for the fermentation of the different strains studied.
- Figure 3 Comparison of total sugars measured at the end of fermentation for the different strains studied.
- Example 1 Obtaining and Characterization of Transformants of Strain 1-4997 containing 1 or 4 copies of a Gene coding for Aspergillus niger Xylanase (XYN1) or of a gene coding for Trichoderma reesei Xylanase (XYN2)
- the strategy implemented to clone a xylanase activity in the strain of Saccharomyces cerevisiae ER-GAND-8159-C1 is based on the use of a multi-integrative expression system. With this system, it is possible to simultaneously integrate one or more copies of a gene encoding a given xylanase at a given locus.
- the inventors chose to integrate 1 copy or 4 copies of one of the two genes XYN1 from Aspergillus niger and XYN2 from Trichoderma reesei in order to measure a possible effect linked to the number of copies on xylanase activity.
- Aspergillus niger xylanase Aspergillus niger xylanase (XYN1).
- the XYN1 gene which codes for AspergiUus niger xylanase was previously amplified by PCR using genetic material derived from the AspergiUus niger strain ATCC10577.
- the PCR product was then cloned into an expression vector (developed internally) under the dependence of a strong promoter pADH1 and the terminator tCYCl.
- the plasmid obtained serves as a matrix for the generation of the expression modules, as described in paragraph B below.
- Trichoderma reesei xylanase (XYN2).
- the Trichoderma reesei xylanase which has been used is encoded by the XYN2 gene.
- the sequence used is the cDNA version, freed of its introns and optimized through codons to improve the translation of the protein in the yeast Saccharomyces cerevisiae.
- the plasmid which has the XYN2 gene dependent on the pADH1 / tCYCl pair, was used as a PCR matrix to synthesize the expression modules.
- the strategy employed by the inventors consisted in simultaneously integrating several expression modules of the xylanase genes in a strain of Saccharomyces cerevisiae in a single step at a given locus, based on the natural capacity of the yeast to carry out homologous recombination in vivo.
- the inventors defined the PCR primers to be used to integrate the modules at the BUD5 locus.
- 1 or 4 expression modules for A niger xylanase or T. reesei xylanase, as well as a selection module have been integrated.
- Each amplified module has recombinogenic sequences (Al, Bl, Cl and Dl) on either side of its promoter and its terminator. These sequences are provided by the floating tails of the PCR primers (Table 1) and allow the modules to align and recombine specifically by homology between these recombinogenic sequences.
- sequences homologous to a given locus for example the BUD5 locus, at the 5 ′ and 3 ′ ends of the multi-integrative expression cassette allows the simultaneous integration of the expression modules and of the selection module by homologous recombination at this given locus.
- the selection of the clones having correctly integrated the expression modules is carried out initially on the basis of the presence of the selection module in the integration cassette.
- the selection module includes a strong promoter / terminator pair and a gene whose expression confers on the yeasts which contain it a characteristic making it possible to select them on a given medium. The inventors have thus isolated the clones resulting from each transformation. Table 1: Listing of Primers Used and Nomenclature of Synthetic Expression Modules.
- Table 2 Mixing of modules before transformation of the strain 1-4997.
- Solid Phenotypic Test In this test, 5 ⁇ L of culture supernatant (YPG 5%, 30 ° C., 24 hours, 150 rpm) were deposited on a medium containing birch xylan, and the halos of xylan hydrolysis were visualized after staining with Congo red 1% and discoloration with 1 M NaCl. Hydrolysis halos were observed for all the clones considered).
- Liquid Phenotypic Test This phenotypic characteristic is based on the same principle as that used for the selection of transformants (see above).
- ER-GAND-8159 is the strain deposited at the CNCM under the number 1-4997.
- Ethanol Red® is a strain deposited at the CNCM on September 4, 2008, by this Applicant, under the number 1-4071.
- the “Alcohol Max” medium contains: 280 g / kg of sucrose, 5 g / kg of yeast extract, 4.7 g / kg of di-basic ammonium phosphate (DAP), 11.5 g / kg of citric acid, 13.5 g / kg sodium citrate, as well as minerals and vitamins.
- DAP di-basic ammonium phosphate
- the transformants were evaluated in a dextrin medium.
- the strain used as host to integrate the expression modules of the xylanases corresponds to the strain ER-GAND-8159 (CNCM 1-4997) which has 2 genes of different origin from glucoamylase
- the dextrins being molecules resulting from l hydrolysis of starch, the clones secreting glucoamylases are capable of degrading them into glucose and thus of producing ethanol.
- the fermentation conditions used were identical to those used with the YFAM medium.
- dextrin medium is meant a synthetic medium containing dextrins, as known to a person skilled in the art. It is for example a synthetic medium containing starch dextrins (220 g / kg), yeast extract (5 g / kg), urea (2 g / kg), dihydrogen phosphate potassium (1 g / kg) as well as minerals and vitamins.
- transformants of the ER-GAND-8159 strain have 1 copy or 4 copies of the Aspergillus niger or Trichoderma reesei xylanase genes, using the one-step multi-copy integration strategy by obtaining specifically designed expression modules.
- the inventors endeavored to validate, by PCR, the genotype of the transformants obtained as well as their phenotype of xylan hydrolysis, and they ensured that their capacities for ethanol production and for hydrolysis of l starch had not been negatively impacted by the genetic modifications made.
- Table 4 Composition of Sugars Available in the Liquefied Substrate.
- the liquefied substrate has a total glucose potential measured by the enzymatic method of 226 g giU co S e / kg su b s trat ⁇
- Yeast Creams from Strains Selected for Evaluation were cultivated on a Petri dish for 24 hours and then stored in the refrigerator before use. Each strain was then removed and used to seed 100 ml of acid medium (for example YM medium) in 250 ml flasks. The flasks were placed in an incubator at a temperature of 26 ° C for 24 hours. At the end of this incubation, each medium was placed in centrifugation at 4500 rpm for 5 minutes. After removal of the supernatant, 150 ml of sterile water were added to wash the yeasts.
- acid medium for example YM medium
- Figure 2 (A) shows the fermentative kinetics observed. It appears that with the exception of the strain ER-GAND-XTR-1C-c18 which exhibits a delay in the initial kinetics, all the strains exhibit a similar start of fermentation during the first 36 hours of fermentation. After 36 hours, the reference strain ER-GAND-8159 slows down, as does the strain ER-GAND-XTR-1C-cl3 (I-5265) whose kinetics are identical. The two strains expressing the xylanase gene of Asp ergillus niger continue the fermentation at a higher speed and produce a greater amount of C0 2 during the test. The ER-GAND-XTR-lC-cl8 strain which had an initial kinetic delay catches up with the reference at 54 hours of fermentation and exceeds it to finish slightly behind compared to the two ER-GAND-XAN strains.
- Figure 2 (B) illustrates the values of ethanol, glycerol / ethanol mass ratio as well as the ethanol yield calculated on the glucose potential of the substrate used.
- Table 5 below presents all of the data collected during the tests to compare performance with the benchmark.
- the left part of the table presents the raw values and the right part presents the gain observed in relative to the reference.
- the strains showing a gain in ethanol production therefore exhibit a reduction in the production of glycerol in parallel.
- this reduction in glycerol does not explain the gain in alcohol; the gain in yield comes from a more efficient consumption of glucose from the medium made possible by the action of xylanase produced by each strain.
- This result is confirmed by the measurements of total sugars at the end of fermentation presented in Ligure 3: the strains with an advantage over their ethanol yield also have a reduced residual sugar level.
- the action of xylanase on the fermentation matrix is beneficial to the action of glucoamylases by allowing them better access to the starch in the medium while reducing the glycerol response of the strains.
- This double gain in sugar leads to better production of ethanol.
- the ER-GAND-XTR-lc strain has a similar advantage, although slightly less in this example.
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FR1856080A FR3083245B1 (en) | 2018-07-02 | 2018-07-02 | STRAIN OF SACCHAROMYCES CEREVISIAE EXPRESSING EXOGENOUS GLUCOAMYLASE AND XYLANASE ENZYMES AND THEIR USE IN THE PRODUCTION OF BIOETHANOL |
PCT/EP2019/067668 WO2020007823A1 (en) | 2018-07-02 | 2019-07-02 | Saccharomyces cerevisiae strains expressing glucoamylase enzymes and exogenous xylanase and their use in the production of bioethanol |
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